The present invention relates to a method for culturing cells derived from adipose tissue, and in particular from the stromal vascular fraction (SVF), to induce the formation of cardiomyocytes.
The subject of the present invention is also the use of the cells obtained by means of the culture method as defined above for reconstituting a cardiac region that has been rendered ischemic, in particular after an infarction, and also a pharmaceutical composition containing said cells.
In many cases, heart failure develops following an ischemic event (myocardial infarction) and is associated with a considerable loss of cardiomyocytes and with ischemia, which is not counterbalanced by the normal renewal of differentiated cardiomyocytes by the heart.
The means that exist for reconstituting cardiomyocytes are essentially based on strategies including a cell therapy, in order to improve the defective contractile performance and cardiac function (Wollert et al., Circulation, 2005, 112, 2, 151-153).
They essentially comprise:                the transplantation of autologous myoblasts or of satellite cells, which improve cardiac function, even though the cells maintain their characteristics of muscle cells in the ischemic heart. Nevertheless, the apparent absence of connections between the grafted myoblasts and the resident cardiomyocytes excludes a synchronized contribution by the grafted cells to the function of the systolic pump;        the concept of cell plasticity has opened up new perspectives in the field of heart failure and has resulted in various cells being used in the hope that these ectopic cells can transdifferentiate into cardiomyocytes, when they are placed in an appropriate in vivo context. In fact, initial studies have suggested that mesenchymal stem cells from the bone marrow and hematopoetic cells can transdifferentiate into cardiomyocytes; however, this phenomenon is currently disputed as is the concept of cell plasticity;        another approach, which is less complicated, has been recommended and proposes to use cells involved in the cardiomyocyte transformation process, such as resident cardiomyocyte stem cells, fetal cells, embryonic cells or other cells such as bone marrow mesenchymal cells or endothelial cells (Kehat et al., J. Clin. Invest., 2001, 108, 407-444; Muller et al., FASEB J., 2000, 14, 2540-2558; Toma et al., Circulation, 2002, 105, 93-98; Liechty et al., Nat. Medecine, 2000, 11, 1282-1286; Conderelli et al., PNAS, 2001, 98, 10733-10738; Wang et al., J. Thorac. Cardiovasc. Surg 2001, 122, 699-705; Jackson et al., J. Clin. Invest. 2001, 107, 1395-1402). Two hypotheses have been put forward to explain these results: cells close to the totipotence of embryonic cells persist in adult tissues (brain, muscle, etc.) and are capable of differentiating into various cell types, or else specialized stem cells of these tissues possess great plasticity and are capable of dedifferentiating or being reprogrammed (transdifferentiation). These results have important consequences for the treatment of functional muscle defects (myopathies and cardiomyopathies) and diseases related to muscle degeneration (myocardium infarction). However, in practice, the effective reconstruction of cardiac muscle tissues from the abovementioned cells is difficult to carry out owing to the technical difficulties in sampling and the small amounts of tissues available. In addition to these technical difficulties, there are also ethical problems associated with the use of embryonic tissues (M/S, 2004, 6-7, 20, 651-661).        
In this context, there exists a real need for new means and in particular for new sources of cells capable of effectively reconstructing the myocardium, which are effective and simpler to implement than the existing means.
Consequently, the inventors gave themselves the aim of providing cells capable of reconstituting cardiac muscle tissues in a long-lasting manner, the cells being isolated from tissues that are easy to sample and available in large amounts.
In the context of their previous research, the inventors have found (application WO 02/055678) that spontaneous differentiation of cells derived from adipose tissue, and more particularly of cells of the stromal vascular fraction (SVF), into functional cardiomyocytes exists when these cells derived from adipose tissue are cultured in a medium containing methylcellulose (PCT International application WO 02/055678; Planat et al., Circ. Res., 2004, 94, 223-229).
Thus, PCT International application WO 02/055678 recommends, in order to obtain differentiation of SVF cells into cardiogenic cells, a step of preparing the SVF, a cell-sorting step (selection) comprising culturing the cells on a semi-solid medium (methylcellulose) and/or purifying the cells by physical separation and immunoselection (antibodies) and a step for “expanding” the cells in DMEM-F12 liquid medium. However, it is found that culturing in a DMEM-F12 liquid medium in fact only makes it possible to maintain the cardiomyogenic cells and not to expand them (Planat-Bénard et al., Circ. Res., 2004, 94, 223-229).
In fact, the abovementioned article in the names of Planat-Bénard et al. shows that the cells of the SVF are capable of spontaneously differentiating into cardiomyocytes when they are cultured in a medium containing methylcellulose. It is also specified that these cells can be maintained for several months on methylcellulose or in a DMEM-F12 liquid culture medium.
Thus, this article shows that cells of cardiomyocyte type can be obtained spontaneously in primary culture from the SVF when a semi-solid medium based on methylcellulose is used as culture medium. On the other hand, when the DMEM-F12 liquid medium is used, very few contractile cells are observed.
Thus, although both the method described in PCT International application WO 02/055678 and that recommended in the abovementioned article in the names of Planat-Bénard et al. make it possible to effectively obtain cardiomyocytes, they do not give a good differentiation yield. This is because the frequency of obtaining cardiomyocytes both in the method described in PCT International application WO 02/055678 and in the article in the names of Planat-Bénard et al. is very low; this is in particular due to the heterogeneity of the SVF cells and to the variability in their preparation, which do not make it possible to obtain, under the standard culture conditions described above, a good yield in terms of differentiation of these cells into cardiac cells.